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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Definitions

• the present disclosure relates to multi-specific molecules which are capable of simultaneously binding at least two different target antigens or epitopes.
• the molecules comprise at least one binding domain molecule (BDM) which binds to a first target antigen or epitope, the BDM being modified for selective binding to a heterologous target, coupled to a pharmacologically active protein or peptide which is an antibody or antigen-binding fragment thereof or a non-antibody protein or peptide which binds to a second target antigen or epitope, the BDMs being coupled to a C-terminus of a polypeptide present within the pharmacologically active protein or peptide.
• BDM binding domain molecule
• proteins in their unmodified form are known to be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticulendothelial system, or proteolytic degradation (Francis (1992) Focus on Growth Factors 3:4-1 1 ).
• Various modifications of proteins and peptides have been developed to increase the therapeutic protein's stability, circulation time and biological activity (see Francis (1 992) Focus on Growth Factors 3:4-10).
• Francis (1 992) Focus on Growth Factors 3:4-10 there exists a need in the art for mechanisms which allow such therapeutic proteins to subsist in vivo for longer.
• antibody products Therapeutic monoclonal antibodies and antibody-related products such as antibody- fusion proteins, antibody fragments, and antibody-drug conjugates (collectively referred to hereafter as antibody products) have grown to become the dominant product class within the biopharmaceutical market.
• Antibody products today are approved for the treatment of a variety of diseases, including some cancers, multiple sclerosis, asthma and rheumatoid arthritis to name but a few.
• antibody like scaffolds are generally constructed from fragments of antibodies or made from antibody like proteins that, like antibodies, can bind to specific targets.
• Dual specificity antibodies allow for more potent antibody drugs which can be designed to redirect and activate immune effector cells such as T-cells to specifically kill tumours; bind to multiple targets and effect multiple pathogenic pathways; bind to multiple sites on the one target cell or protein to increase specificity or induce synergistic induction; and target tumours that are heterogeneous in nature.
• bi-specific has a number of significant drawbacks: Firstly, the small size of bi-specifics created by fusing two or more antibody-like scaffolds together, are generally significantly below the renal threshold and typically show very short blood circulation half-life which is in the range of minutes to hours. Such a short half-life necessitates dosing intervals of every day or via constant infusion which can lead to exceeding the toxicity threshold of the drug. And secondly, the affinity of many antibody-like scaffolds for their target is inadequate due to their monovalent nature (i.e. they have only one antigen binding site compared to antibodies which have two). Full antibodies bind with both antigen binding sites improving the overall strength of binding (knows as an avidity effect) giving a certain advantage compared with monovalent antibody-like scaffolds.
• the present disclosure is based on approaches to improve one or more characteristics of a protein or peptide, including an antibody or an immunoglobulin antigen-binding fragment. More particularly, the present disclosure is based on approaches that improve a poorly therapeutic protein (e.g. therapeutic antibody) by converting it into a multi-specific format. Coupling the protein or peptide to at least one binding domain molecule (BDM) described herein provides a bi-specific or multi-specific molecule thus allowing the molecule to bind to different targets by virtue of exploiting the different binding targets (i.e. antigens or epitopes) of the protein and the BDM. Accordingly, one or more characteristics of the protein or peptide can be improved including therapeutic efficacy, half-life, immune engagement, avidity, cellular penetration and/or tolerability. The molecules thus provide an alternative to traditional bi- specific antibodies.
• BDM binding domain molecule
• the target antigen or epitope bound by the BDM is different to the target antigen or epitope bound by the protein or peptide.
• the protein may bind to a target antigen or epitope present on a cell or tissue and the BDM may bind to a target antigen or epitope present on an immune-modulating cell such as a cytotoxic T cell or protein to facilitate cell killing, or a target antigen on human serum albumin (HSA) so that the half-life of the protein or peptide may be extended.
• an immune-modulating cell such as a cytotoxic T cell or protein to facilitate cell killing, or a target antigen on human serum albumin (HSA) so that the half-life of the protein or peptide may be extended.
• HSA human serum albumin
• the therapeutic efficacy of the protein or peptide can be promoted by utilising the functionality of the target to which the BDM binds.
• the protein or peptide can be converted into bi-specific, tri-specifics or even multi- specifics.
• the smaller size, binding affinity characteristics and solubility of the BDMs make them ideal agents for improving the efficacy of poorly therapeutic proteins or peptides, for example, facilitating the body's natural immunological mechanism for destroying tumour cells.
• the present disclosure thus provides a multi-specific molecule capable of binding to two or more different target antigens or epitopes, the molecule comprising: (i) at least one binding domain molecule (BDM) which binds to a first target antigen or epitope, the BDM comprising or consisting of a V-like domain (VLD) scaffold having three exposed binding loops (BLs) contained within and wherein at least two of the three BLs are modified or replaced relative to their corresponding native sequence in the scaffold for selective binding to a heterologous target antigen or epitope; and
• BDM binding domain molecule
• VLD V-like domain
• a pharmacologically active protein or peptide which is an antibody or antigen-biding fragment thereof or a non-antibody protein or peptide which binds to a second target antigen or epitope;
• the pharmacologically active protein binds to its native target antigen or epitope.
• the epitopes are on separate antigens.
• the first and second target antigens are different. In one example, the first and second target antigens are the same but the molecule binds to different epitopes on the target antigen. In one example, the first and second target epitopes are different.
• the molecule comprises least two BDMs, or at least one pair of BDMs, wherein each BDM (or BDM pair) binds to a different target antigen or epitope.
• each BDM (or BDM pair) binds to a target antigen or epitope that is different from the target antigen or epitope to which the pharmacologically active protein or peptide binds.
• the non-antibody protein or peptide is selected from the group consisting of a blood clotting factor, an anticalin, a toxoid, a collagen binding protein, a human serum binding protein (e.g. Human serum albumin, HSA) a tumour necrosis factor (TNF)-alpha receptor binding protein, an integrin binding protein, a vascular endothelial growth factor (VEGF) or mimetic thereof, an erythropoietin (EPO) or mimetic thereof, a C4 binding protein, a urokinase receptor antagonist, a lymphokine, a cytokine, an osteoprotegerin (OPG), or the extracellular domain of a protein selected from programmed cell death 1 protein (PD1 ), programmed death ligand 1 (PD-L1 ), NKG2D, MHC class I polypeptide related sequence A (MICA), MHC class I polypeptide related sequence B (MICB), UL16 binding protein (ULBP).
• the blood clotting factor is factor VIII or factor IX.
• the at least one BDM is coupled to a C-terminus of an antibody heavy chain polypeptide. In one example, the at least one BDM is coupled to a C-terminus of both antibody heavy chain polypeptides.
• At least one BDM is coupled to a C-terminus of the constant region of a light chain polypeptide of an Fab
• BL1 and BL3 are modified or replaced from the native BL sequence. In another example, BL1 , BL2 and BL3 are modified or replaced from the native BL sequence.
• the BDM scaffold comprises or consists of the whole or part thereof of a native Ig-like domain or an Ig-like domain with altered binding loops (i.e. modified Ig-like domain) relative to the native Ig-like domain, wherein the Ig-like domain is selected from the group consisting of a ThyOx family member polypeptide, a T cell receptor, CD2, CD4, CD8, class I MHC, class II MHC, CD1 , cytokine receptor, G-CSF receptor, GM-CSF receptor, hormone receptors, growth hormone receptor, erythropoietin receptor, interferon gamma receptor, prolactin receptor, NCAM, VCAM, ICAM, N-caderin, E-caderin, fibronectin, tenascin, and l-set containing domain polypeptides or a functional fragment thereof.
• a ThyOx family member polypeptide a T cell receptor
• CD2, CD4, CD8 class I MHC, class II MHC, CD1
• a VLD or C-set domain may encompass a BDM which binds to a heterologous target antigen or epitope.
• a modified VLD or C-set domain may also comprise one or more modifications which alter the affinity of the BDM to its native target. The affinity towards the native target may be increased or decreased compared to the native VLD or C-set domain.
• the BDM comprises or consists of the whole or part thereof of a VLD protein or C-set domain protein comprising between 5 and 30 amino acid substitutions, between 5 and 20 amino acid substitutions, between 5 and 15 amino acid substitutions, between 5 and 10 amino acid substitutions, or up to 5 amino acid substitutions compared to the corresponding native VLD or C-set domain protein.
• the BDM is not the CLTA-4 VLD mutant molecule L104EA29Y or L104E described in US 7,094,874.
• the modified BDM comprises one heterologous BL sequence. In another example, the modified BDM comprises two heterologous BL sequences. In yet another example, the BDM comprises three heterologous BL sequences.
• amino acid residues at positions 27 to 33, and/or positions 54 to 62 and/or positions 98 to106 of SEQ ID NO:1 are modified or replaced with heterologous sequence.
• the BDM VLD scaffold comprises or consists of the sequence
• the BDM VLD comprises or consists of the sequence KAMHVAQPAVVLASSRGIASFVCEYTVSWVDMEVRVTVLRQADSQVTEVCAATYWNGRWLT FLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVQLDPSWGYYWQGYEGIGNGTQIYVIDPE PSPDSN (SEQ ID NO:14), wherein the BDM binds to sclerostin.
• BL-1 , BL-2 and BL-3 of the BDM are replaced with the CDR1 , CDR2 and CDR3 sequences respectively of an antibody.
• the antibody from which the CDR sequences are derived may be derived from any species.
• the antibody is derived from a human.
• the antibody is derived from a domestic animal, for example, cat, dog, rabbit, guinea pig or horse.
• Linking of BDM monomers may be achieved for example by use of covalent or non- covalent bonds or by use of a short peptide linker as described further herein. Any of the linking methodologies referred to herein can be employed to link the BDM monomers together. Alternatively adjacent BDM monomers may be directly fused together.
• coupling of the pharmacologically active protein and the at least one BDM is achieved without use of a linker.
• the molecule is capable of simultaneous binding to B7-1 -Fc and sclerostin.
• the present disclosure also provides a polypeptide selected from the group comprising or consisting of a sequence of any one of SEQ ID NOs: 5, 6, 13, 14, 19, 21 , 22, 23, 24, 25, 27, 28 or 29.
• the polypeptide is isolated.
• a polypeptide of the present disclosure includes a polypeptide tag. Examples, of suitable tags include, but are not limited to the p97 molecule, myc, hexa-his tag, flag, E7.
• the molecule according to the disclosure is a nucleic acid.
• N1 is length of nucleotides encoding a first binding loop
• N2 is a length of nucleotides encoding a second binding loop
• N3 is a length of nucleotides encoding a third binding loop.
• N1 , N2 and N2 are between 15 and 45 nucleotides.
• N1 is between 15 and 24 nucleotides.
• N2 is 15 nucleotides and N3 is between 30 and 45 nucleotides.
• N is any nucleotide (A, C, T, G).
• the nucleic acid may further include a moiety e.g. FLAG to facilitate purification and identification.
• a moiety e.g. FLAG to facilitate purification and identification.
• the present disclosure also provides a method for producing the polypeptide molecule of the present disclosure comprising culturing the host cell of the present disclosure under conditions enabling expression of the polypeptide and optionally recovering the polypeptide.
• the polypeptide may be glycosylated or unglycosylated.
• the antibody nucleic acid sequence may further comprise the hinge region.
• B7-1 -Fc was replaced with Buffer at Point 2.
• Trace 3 is the bi-specific - D1 .3 lgG-VLDx2 (LC) binding to lysozyme immobilised on the biosensor surface followed with an addition of buffer at Point 1 .
• Trace 4 is the bi-specific - D1 .3 lgG-VLDx2 (LC) binding to lysozyme immobilised on the biosensor surface followed by an addition of B7-1 -Fc at Point 1 .
• B7-1 -Fc was replaced with Buffer at Point 2.
• the sensorgram demonstrates simultaneous, dual target binding to lysozyme and B7-1 -Fc.
• Figure 22 shows a series of SPR binding sensorgrams that have been overlaid demonstrating initial binding of the tri-specific [IgG VLDx4 (Scl-HC)(B7-LC)] to streptavidin captured biotin labelled lysozyme followed by sequential and simultaneous binding to B7-1 -Fc and sclerostin.
• the tri-specific has sclerostin (Scl) binding VLDs fused to the D1 .3 antibody [D1 .3 IgG] heavy chains and B7-1 binding VLD's fused to the light chains.
• Buffer Added Point at which sclerostin is replaced with buffer.
• immunoglobulin antigen-binding fragment as used herein is intended to refers to a fragment of an antibody, which fragment includes a light chain variable region and a heavy chain variable region having complementarity determining regions (CDRs).
• CDRs complementarity determining regions
• the term encompasses an Fab, F(ab') 2 , Fab', scFv, di-scFv, or chemically linked F(ab') 2 .
• the term 'epitope' (syn. "antigenic determinant”) shall be understood to mean a region to which a protein (or an antigen-binding domain of an antibody or immunoglobulin antigen-binding fragment) binds or which the BDM of the present disclosure binds.
• the term refers to a structure bound by an immunoglobulin VH/VL pair.
• An epitope defines the minimum binding site for an antibody or antibody-like domain (e.g. BDM). This term is not necessarily limited to the specific residues or structure to which a protein and/or the BDM of the molecule makes contact.
• target refers to an antigen or an epitope.
• the target refers to a cell-surface protein e.g. receptor or a viral coat protein.
• the target is a secreted protein.
• the term 'antigen binding domain' in the context of an antibody or immunoglobulin antigen-binding fragment shall be taken to mean a region of an antibody or immunoglobulin antigen-binding fragment that is capable of specifically binding to an antigen, more particularly an epitope present on an antigen.
• the antigen binding domain corresponds to the V H and V L . Within the V H and V L regions are the CDRs which make contact with the epitope.
• peptide as used herein is taken to refer to a short chain (typically of about 50 amino acids or less) of amino acid monomers linked by peptide (amide) bonds.
• a BDM scaffold of the present disclosure preferably exhibits less than about 50% amino acid identity to a human immunoglobulin variable heavy or light chain sequence.
• the scaffold will exhibit, for example, amino acid sequence identity less than about 45%, about 40%, about 30%, about 20%, about 15%, or about 10% compared to a human immunoglobulin variable heavy or light chain amino acid sequence.
• VLDs are typically distinguished from those of antibodies or T-cell receptors because they have no propensity to join together into Fv-type molecules. VLD are discussed in The Leucocyte Antigen Facts Book 1993, Eds Barclay et al., Academic Press, London; and in CD Antigens 1996 (1997) Immunology Today 1 8, 100-101 , and in Arlene H Sharpe and Gordon J Freeman, (2002) Nature Reviews Immunology 2, 1 16-126, the entire contents of which are incorporated herein by reference. A person skilled in the art in the subject matter of the present disclosure can readily determine appropriate VLD containing proteins suitable for use herein for example by conducting a search of the Uniprot database (www.uniprot.org).
• CTLA-4 Cytotoxic T-lymphocyte associated antigen 4
• CD28 and ICOS are involved in T-cell regulation during the immune response.
• CTLA-4 is a 44 kDa homodimer expressed primarily and transiently on the surface of activated T-cells, where it interacts with CD80 and CD86 surface antigens on antigen presenting cells to effect regulation of the immune response (Waterhouse et al. (1996) Immunol Rev 153:183-207, van der Merwe et al. (1997) J Exp Med 185(3):393-403).
• Each CTLA-4 monomeric subunit consists of an N-terminal extracellular domain, transmembrane region and C-terminal intracellular domain.
• the extracellular domain comprises an N-terminal V-like domain (VLD; of approximately 14 kDa predicted molecular weight by homology to the immunoglobulin superfamily) and a stalk of about 10 residues connecting the VLD to the transmembrane region.
• VLD comprises surface loops corresponding to BL-1 , BL-2 and BL-3 respectively (Metzler WJ et al (1997) Nat Struct Biol 4(7):527-31 ) which binds to CD80 and/or CD86.
• the sequence of human CTLA-4 has been previously determined (US 5,434,131 ; US 5,844,095; US 5,851 ,795).
• CTLA-4 The human sequence for CTLA-4 is available as UniProt reference P1 6410.
• the extracellular domain of CTLA-4 corresponds to positions 36-161 of the sequence (wherein the CTLA-4 has a total length of 126 amino acids).
• Amino acid residues 1 -35 correspond to the signal peptide.
• Proteins such as basigin contain a C-set domain (Xiao-Ling Yu et al. (2008) JBC vol 283(26):18056-1 8065). Further examples of C-set domains include ROR1 extracellular domain, CEA family members such as CEACAM1 -8.
• An i-body is a single domain antibody-like molecule of human origin.
• the i-body framework resembles the single domain antibody from sharks and as a result shares the favourable biophysical and targeting properties of the shark antibody. They are described in for example, US 7,977,071 .
• a VNAR variant new antigen receptor refers to a single variable region domain fragment derived from a shark immunoglobulin new antigen receptor antibody (IgNAR). They are described for example in Griffiths K et al (2013) Antibodies 2(1 ):66-81 .
• the molecule of the present disclosure may bind to at least one target antigen, at least two different target antigens, at least three different target antigens, at least four different target antigens or at least five different target antigens.
• the protein or peptide binds to a first target antigen which is the same or different as the second target antigen bound by the BDM;
• the framework residues of the BDM may be modified in accordance with structural features present in camelid antibodies.
• the camel heavy chain immunoglobulins differ from conventional antibody structures by consisting of a single VH domain.
• cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulphide bond formation in this region.
• the antibody thus generated can have improved internalisation capability and/or increased complement mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., (1992) J. Exp Med., 176: 1 191 -1 1 95 and Shopes, (1992) J. Immunol., 148: 2918-2922.
• ADCC antibody-dependent cellular cytotoxicity
• the present disclosure also provides methods for making a multi-specific molecule of the present disclosure.
• the term 'promoter' is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner.
• promoter is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked.
• Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.
• Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EF1 ), small nuclear RNA promoters (U1 a and U1 b), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, ⁇ -actin promoter; hybrid regulatory element comprising a CMV enhancer/ ⁇ -actin promoter or an immunoglobulin promoter or active fragment thereof.
• CMV-IE cytomegalovirus immediate early promoter
• EF1 human elongation factor 1 -a promoter
• U1 a and U1 b small nuclear RNA promoters
• a-myosin heavy chain promoter Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, ⁇ -actin promote
• the present disclosure also provides a method of producing a multi-specific molecule of the present disclosure which method comprises the steps of:
• the method comprises:
• fusion or linkage between a protein (e.g. antibody) and a BDM may be achieved by conventional covalent or ionic bonds, protein fusions, or heterobifunctional cross- linkers, e.g., carbodiimide, glutaraldehyde, and the like.
• Conventional inert linker sequences e.g. peptide linkers
• the design of such linkers is well known to those of skill in the art and is described for example in US 8,580,922; US 5,525,491 ; and US 6,165,476.
• the linker can facilitate enhanced flexibility, and/or reduce steric hindrance between any two proteins.
• the linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein.
• An exemplary linker sequence is the linker found between the C-terminal and N-terminal domains of the RNA polymerase alpha subunit.
• Other examples of naturally occurring linkers include linkers found in the 1 CI and LexA proteins.
• the amino acid sequence may be varied based on the preferred characteristics of the linker as determined empirically or as revealed by modelling. Considerations in choosing a linker include flexibility of the linker, charge of the linker, and presence of some amino acids of the linker in the naturally-occurring subunits.
• the linker can also be designed such that residues in the linker contact DNA, thereby influencing binding affinity or specificity, or to interact with other proteins. In some cases, particularly when it is necessary to span a longer distance between subunits or when the domains must be held in a particular configuration, the linker may optionally contain an additional folded domain.
• Non-peptide linkers are also possible.
• These alkyl linkers may be further substituted by any non-sterically hindering group such as lower alkyl (e.g. Ci-C 6 ), lower acyl, halogen (e.g. CI, Br), CN, NH 2 , phenyl.
• An exemplary non-peptide linker is a PEG linker having a molecular weight of 100 to 5000kD, preferably 100 to 500kD.
• linkers which are suitable for use include GSTVAAPS, TVAAPSGS or GSTVAAPSGS or multiples of such linkers.
• Binding of epitopes can be measured by conventional by conventional antigen binding assays, such as ELISA, by fluorescence based techniques, including FRET, or by techniques such as surface plasmon resonance which measure the mass of molecules.
• Specific binding of an antigen binding protein (e.g. BDM) to an antigen or epitope can be determined by suitable assay, including, for example, Scatchard analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassays such as ELISA and sandwich competition assays.
• suitable assay including, for example, Scatchard analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassays such as ELISA and sandwich competition assays.
• Affinity can be at least 1 -fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 1 0-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1 000-fold greater, or more, than the affinity of the protein or the BDM for unrelated amino acid sequences.
• Affinity of a protein or BDM to a target e.g.
• the nucleic acid target may be DNA, RNA or a combination of DNA and RNA.
• the multi-specific molecules of the present disclosure can be used as a composition when combined with a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for administration to a subject in vivo.
• compositions can be formulated to be compatible with a particular route of administration, systemic or local.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• label or labelled is intended to encompass direct labelling of the protein (e.g. antibody) or BDM by coupling coupling (i.e. physically linking) a detectable substance to said protein or BDM, as well as indirect labelling by reactivity with another reagent that is directly labelled.
• label also includes covalent or non-covalent coupling.
• the molecule can be labelled with a toxin, a radionuclide, iron-related compound, a dye, an imaging agent or a fluorescent label or a chemotherapeutic agent.
• taxanes such as paclitaxel and docetaxel
• nitrogen such as mechlorethamine, melphalan, uracil mustard and chlorambucil
• ethylenimine derivatives such as thiotepa
• alkyl sulfonates such as busulfan
• nitrosoureas such as lomustine, semustine and streptozocin
• triazenes such as dacarbazine
• folic acid analogs such as methotrexate
• pyrimidine analogs such as fluorouracil, cytarabine and azaribine
• purine analogs such as mercaptopurine and thioguanine
• vinca alkaloids such as vinblastine and vincristine
• antibiotics such as dactinomycin, daunorubicin, doxorubicin, and mitomycin
• enzymes platinum coordination complexes, such as cisplatin
• substituted urea such as
• the present disclosure also provides a method for detecting a target to which either or both of the protein and BDM moieties of the polypeptide bind.
• Such methods may for example, employ the use of detectable labels as described above.
• different labels could be utilised for the protein and the BDM to identify whether the target bound is that bound by the protein or the BDM.
• target protein can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose.
• the support can then be washed with suitable buffers followed by treatment with the detectably labelled polypeptide.
• the solid phase support can then be washed with the buffer a second time to remove unbound polypeptide.
• the amount of bound label on the solid support can then be detected by conventional means.
• a factor VIII protein can be coupled to a VLD that binds to human serum albumin so that the half-life of the protein is increased. This has obvious advantages in terms of less frequent dosing for subjects with haemophilia.
• the molecules according to the present disclosure provide advantages over therapeutics based on bi-specific antibodies.
• a simple and efficient approach for improving one or more characteristics of an antibody, e.g. poorly therapeutic antibody is to attach one or more single binding domain molecules (BDM) that bind to specific therapeutic targets to an antibody or an immunoglobulin antigen-binding fragment which has specificity to a target of interest.
• BDM single binding domain molecules
• Anti-lysozyme Fab heavy chain coupled to B7.1 binding VLD with 21 aa Gly-Ser linker designated D1 .3 Fab-VLDx1 (HO (SEQ ID NO:23)
• Figure 2 shows a schematic of the antibody-VLD bi-specific molecule according to one example created by coupling of a target specific VLD (e.g. that bind to Target B) to the terminal end of each constant heavy (CH3) chain sequence of the anti-Lysozyme lgG1 antibody heavy chain [D1 .3 lgG-VLDx2 (HC)].
• the antibody molecule binds to target A via the antibody heavy and light chain variable regions and to target B via the VLD on the heavy chain.
• the molecule binds to target A via the antibody heavy and light chain variable regions and to target B via the VLD on the light chain.
• Figure 4 shows a schematic of the antibody-VLD tri-specific molecule according to one example created by coupling of a target specific VLD (e.g. that bind to Target B) to both the heavy and light chains D1 .3 lgG-VLDx4 (LC+HC) where a VLD is coupled to the terminal CL sequence and also to the terminal end of the heavy CH3 sequence.
• the molecule binds to target A via the antibody heavy and light chain variable regions, to target B via the VLD on the light chain and to target C via the same VLD or a different VLD to that which binds target B.
• the tri-specific can bind to Target A, B and C either individually or at the same time or in a combination of the three targets (e.g. Target and B or Target A and C or Target B and C).
• the antibody or Fab binds to lysozyme and the VLD binds to B7.1 .
• the parent Fab is D1 .3 Fab
• the bi-specific are D1 .3 Fab-VLDx1 (HC) which is the D1 .3 Fab plus a VLD fused to the terminal end of the CH sequence of the D1 .3 Fab
• D1 .3 Fab-VLDx1 (CL) which is the D1 .3 Fab plus a VLD fused to the terminal end of the CL sequence of the D1 .3 Fab.
• the tri-specific is D1 .3 Fab-VLDx2 (HC+LC) which is the D1 .3 Fab plus VLDs fused to both the terminal end of the CH sequence and CL sequence of the D1 .3 Fab. Results are shown in Figure 8 under non- reducing conditions. Results are known in Figure 9 under reducing conditions. Analysis indicated that appropriate heavy and light chain fusions were at the size ranges expected.
• Example 3 Assessment of binding of bi-specific and tri-specific molecules
• Bi-specific injected is the point at which the IgG VLDx2 (HC) is added to the sensor surface.
• the trace shows the IgG VLDx2 (HC) binding to lysozyme immobilised on the biosensor surface.
• Buffer injected 1 Point at which the injection of IgG VLDx2 (HC) is stopped and replaced with buffer. The trace shows the dissociation of the IgG VLDx2 (HC) from the lysozyme immobilised on the biosensor surface.
• Figure 1 5 shows binding of the bi-specific [IgG VLDx2 (LC)] to streptavidin captured biotin labelled lysozyme followed by secondary binding to a concentration series of B7-1 -Fc (at
• the trace shows the dissociation of B7-1 -Fc from IgG VLDx2 (LC) still attached to the lysozyme immobilised on the biosensor surface.
• Bi-specific Injected Point at which the Fab-VLDx1 (HC) is added to the sensor surface.
• the trace shows the Fab-VLDx1 (HC) binding to lysozyme immobilised on the biosensor surface.
• Buffer Injected 1 Point at which the injection of Fab-VLDx1 (HC) is stopped and replaced with buffer.
• the trace shows the dissociation of the Fab-VLDx1 (HC) from the lysozyme immobilised on the biosensor surface
• B7-1 -Fc Injected Point at which the second analyte B7-1 -Fc is added at specified concentrations (at 25, 12.5, 6.25, 3.125, 1 .56 and 0 ug/ml).
• the trace shows B7-1 -Fc binding to the Fab-VLDx1 (HC) that is still bound to the lysozyme immobilised on the biosensor surface.
• Buffer Injected 1 Point at which the injection of Fab-VLDx1 (LC) is stopped and replaced with buffer. The trace shows the dissociation of the Fab-VLDx1 (LC) from the lysozyme immobilised on the biosensor surface.
• B7-1 -Fc Injected Point at which the second analyte B7-1 -Fc is added at specified concentrations (at 25, 12.5, 6.25, 3.125, 1 .56 and 0 ug/ml). The trace shows B7-1 -Fc binding to the Fab-VLDx1 (LC) that is still bound to the lysozyme immobilised on the biosensor surface.
• Buffer Injected 2 Point at which the injection of B7-1 -Fc is stopped and replaced with buffer. The trace shows the dissociation of B7-1 -Fc from Fab-VLDx1 (LC) still attached to the lysozyme immobilised on the biosensor surface.
• the surface was regenerated by injecting 1 0mM Glycine buffer at pH 2.1 for 30 seconds.
• the Fab VLDx2 (HC+LC) construct was modified whereby a B7-1 binding VLD was coupled to the C terminus of the heavy chain of the anti-lysozyme Fab D3.1 and a sclerostin (Scl) binding VLD was coupled to the Fab constant light (CL) chain of an anti-lysozyme Fab.
• This tri-specific molecule was designated [Fab VLDx2 (B7-1 -HC)(Scl-LC)].
• the biosensor traces show the tri-specifics initially binding to lysozyme immobilised on the biosensor surface followed by binding to B7-1 - Fc (B7-1 -Fc was added at the point indicated).
• the binding traces demonstrate simultaneous, dual target binding to lysozyme and B7-1 -Fc.
• Sclerostin is subsequently added at the point indicated and the biosensor trace shows simultaneous, tri-target binding to lysozyme and B7-1 - Fc and sclerostin for both the Fab VLDx2 (B7-1 -HC)(Scl-LC) and Fab VLDx2 (Scl-HC)(B7-1 - LC) molecules.
• sclerostin is replaced with buffer to show the dissociation rate.
• Figure 25 shows a schematic of a protein coupled to a VLD according to one example of the disclosure.
• the bi-specific can bind to both Target A and B either individually or at the same time.
• Sequences encoding human serum albumin (HSA) fused to one or two VLDs were produced, including a 1 6 amino acid linker sequence (SGGGGSGGGGSGGGGS) highlighted and a C-terminal histidine tag.
• the sequences were cloned into a mammalian expression vector.
• the vector used was the pcDNA3.4 vector (Thermo Fisher).
• the sequences were cloned with a signal peptide to allow the protein to be secreted.
• the sequence of the peptide was as follows MAWMMLLLGLLAYGSG (SEQ ID NO:8).
• Figure 26 shows western blot analysis and detection with anti-His HRP (Sigma Aldrich, Cat # 1 1965085001 ) of the purified HSA fusion proteins, which comprise of VLDs fused to the C-terminus, the N-terminus, or both the C-terminus and N-terminus of HSA.
• the estimated sizes of the proteins, respectively, are: 67 kDa (predicted size: 81 .9 kDa); 69.3 kDa (predicted size: 81 .8 kDa); and 94.4 kDa (predicted size: 96.2 kDa).
• the binding properties of the purified molecules were characterised using the ForteBio Blitz biosensor using standard chemistry and reagents.
• a streptavidin capturing surface (SA Sensor, ForteBio cat# 18-5019) was used to capture biotin labelled protein.
• the HSA-VLD molecule was passed over the target captured on the biosensor surface to generate a binding sensorgram.
• CTLA4 VLD fused to Human Serum albumin at either the C-terminus, N-terminus or both the C and N terminus of HSA.

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